| MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression at post-transcriptional level. The regulation of miRNA on gene expression has been found to be critical for many physiological and pathological processes. Aberrant expression of miRNAs has been observed in many human diseases such as cancers and cardiovascular diseases. There are tremendous interests to develop the expression profile of miRNAs as novel biomarkers for the early detection of cancers and other diseases. MicroRNAs that have been confirmed to be closely related to the occurrence of certain diseases were also considered to be potential therapeutic targets for the development of new drugs. Identification of small molecule that can modulates miRNA expression or function is current research interests as well since the biological active compound identified can be a probe to study miRNA regulatory network at molecular level on one hand, and can be a novel therapeutics for miRNA-related diseases on the other. In this dissertation, we focused on the chemical biological studies on small molecules that are able to regulate endogenous miRNAs, with attempts to elucidate their possible regulatory mechanisms and to explore their biological functions and potential applications. Main work presented in this dissertation was divided into the following three parts: 1. Construction of molecular probe based on the universal inhibitor of miRNAs and the elucidation of its mechanism of action inside cells. From the small molecules screened by cellular assay systems based on Luciferase reporter gene, we identified norathyriol that was derived from natural product mangeferin as effective inhibitor of endogenous miRNAs. To construct a small molecule-probe that was able to universally inhibit miRNAs inside cells, we modified norathyriol by adding bioorthogonal funactionalities on the hydroxyl groups. A series of substituted norathyriol derivatives were obtained among which la and lb with propargyl and allyl respectively substituted on the6-hydroxyl group of norathyriol were found as universal inhibitors of miRNAs. Then the small active molecule la and lb were used as probes to investigate the mechanism of action of this type of small molecule inhibitors. Investigation on the level of miRNAs in the cytoplasm of the compound-treated cells and that associated with the immnoprecipiated AG02proteins revealed that the universal inhibition of la and lb were able to block the association of AG02with endogenous miRNAs. To confirm the interactions of the small molecules with AG02or proteins related to function of miRNAs, bioorthogonal reactions were performed to pull-down proteins associated with la (capable for click reaction) in cells. Purification and identification of the proteins pulled down with the small molecule probe la revealed that AG02was indeed one of the major components with strong interactions with the small-molecule inhibitors. Using click reaction of fluorescent azide with la, we were able to label protein that binds with la inside cells. We observed stronger fluorescence signal from AGO2-overexpressed cells than normal cells. The work presented in this chapter demonstrated the first example of small-molecule universal inhibitor of miRNAs with confirmed interactions with AG02as the mechanism of the universal inhibition. Biological function of the universal inhibitors la and lb to stimulate cancer cells apoptosis was also explored. 2. Investigation of myomiR-involved regulatory pathway using small-molecule selective inhibitor of myomiRs. Based on the cellular assay system with luciferase reporter of various miRNAs, we identified an active molecule compound14from photocycloadducts of2-methoxy-1,4-naphthalenequinone with a series of aryl acetylenes as a selective inhibitor of muscle specific myomiRs (miR-133a, miR-1, miR-206). Then this selective inhibitor of myomiRs was used to treat C2C12cells to investigate its mechanism of action in C2C12cells. QPCR quantification of the expression level of endogenous myomiRs revealed that14was able to significantly down-regulate the myomiRs primary miRNAs expression and also myogenic differentiation markers expressions in myoblast under myogensis condition. These results indicated that compound14might have inhibited a common upstream transcription factor of myomiRs and myogenesis genes. Since the transcriptional factor myoD has been reported to regulate both myomiRs and muscle cell differentiation, we checked the expression of myoD in C2C12cells with and without treatment of compound14. The results showed that mRNA expression of myoD was not downregulated by14, but expression of myoD protein was significantly downregulated in cells treated by the same compound. The suppression of myoD gene translation suggested the possibility of miRNA regulation on the protein expression of the transcriptional factor myoD. Combined with bioinformatic prediction and the use of compound14as a probe to acutely inhibit myoD protein expression, we found that the transcription factor myoD was regulated by miR-221/222and compound14was able to inhibit myoD translation by upregulating miR-221/222expressions. This new miR-221/222-myoD-myomiRs regulatory pathway was further confirmed by conventional miRNA target validation. Herein, a new myoD-involved signaling pathway in C2C12cells was revealed with aid of small molecule inhibitor of myomiRs.3. Identification of a small molecule probe with bioorthogonal functionality and selective inhibition on miR-133a/b and preliminary investigation on its biological function in myogesis and adipogenesis. Myogenic and adipogenic differentiation are of current research interests in life sciences. More and more evidences have suggested the negative regulation of miR-133a on the differentiation of brown adipocytes and dissipation of energy. Therefore we put initial efforts to get a useful small-molecule probe with selective inhibition on miR-133in various cell lines to investigate the miR-133mediated regulatory network. Based on the active molecules we obtained in the previous chapter, we modify the compounds from the photocycloadditions to get an active compound Z that inhibited miR-133a in various cells. Through structure-activity relationship study of Z, we found a suitable modification site that allows biororthogonal group insertion without changing the inhibition activity on myomiR. Then the molecular probe Z’2with a terminal alkyl group for "click" reaction was obtained. Through analyzing effect of Z’2on miR-133in different cells, Z’2was found to have significant inhibitory effect on miR-133a and miR-133b in myoblast (C2C12cells), white preadipocytes (3T3-L1cells) and subcutaneous white preadipocyte. Based on the fact that downregulation of miR-133a/b will lead to upregulation of Prdm-16and consequently induce the transformation of these cells into beige or brown adipocytes, we checked the myogenic and adipogenic selective genes in these cells treated by Z’2. The preliminary results indicated that compound Z’2was able to reprogram these cells to express BAT-selective genes by upregulating Prdm-16expression. Thus this active small molecule has great potential to be developed into a probe for investigating miR-133mediated myogenesis and adipogenesis signaling pathway. With the bio-orthogonal functional group on the small molecule probe, future work on the elucidation of its intracellular target and mechanism of action will be proceeded in our group. |
PDF Full Text Request